Insulin resistance and increased oxidants are characteristics of endothelial cells exposed to a variety of[unreadable] cardiovascular risk factors that contribute to atherosclerosis. The principal hypothesis of this proposal is that[unreadable] oxidants that arise in endothelial cells are themselves responsible for interfering with normal insulin signaling[unreadable] that maintains normal endothelial function. Preliminary studies indicate that exposure of endothelial cells to[unreadable] peroxynitrite (ONOO-) or oxidized LDL interrupts insulin-mediated signaling via PIS kinase and Akt, which[unreadable] normally phosphorylates and stimulates NO release from endothelial nitric oxide synthase (eNOS). The[unreadable] purpose of the proposed studies is to investigate the molecular mechanism of this phenomenon and its[unreadable] consequences with respect to aortic endothelial dysfunction and atherosclerosis. Endothelium-derived oxidants[unreadable] exert specific effects on cell signaling. Our preliminary studies indicate that ONOO- reacts with redox-sensitive[unreadable] thiols on the small GTPase, Ras, thereby activating the protein and initiating downstream signaling in cultured[unreadable] bovine aortic endothelial cells (BAEC). We propose to study these novel redox-dependent effects on Ras and[unreadable] its role in mediating insulin resistance and endothelial dysfunction. We will use proteomic methods to[unreadable] determine the mechanisms by which oxidants influence Ras activity, and also to screen for oxidant changes in[unreadable] other signaling elements that participate in insulin signaling. The significance of oxidant-mediated endothelial[unreadable] insulin resistance in impaired NOS activity, endothelial inflammation, and atherogenesis will be examined in[unreadable] insulin-resistant low density lipoprotein deficient (LDLr -/-) mice fed a high fat, high sucrose (HFHS) diet, and in[unreadable] transgenic mice with conditional endothelial cell specific expression of a Ras dominant-negative, a[unreadable] constitutively active Ras, as well as a constitutively active Akt mutant protein. These studies will elucidate the[unreadable] mechanism by which oxidants impair insulin signaling and determine the importance of impaired signaling to[unreadable] Akt for decreased NO production by eNOS and increased atherogenesis.